Chymosin (EC 3.4.23.4) is the predominant milk-clotting enzyme in the fourth stomach of unweaned calves Use of this enzyme in cheese making has been known since ancient times.
Calf chymosin used for commercial cheese making is typically found in rennet, an impure extract leached from the calf stomach. In calf rennet, 5-20% of the milk clotting activity is due to bovine pepsin with the amount of pepsin in the rennet being a function of the age, weaning and diet of the calves whose stomachs are processed during its preparation. When mixtures of pepsin and chymosin are used to make cheddar cheese, higher levels of non-protein nitrogen and fat are found in the whey when rennet containing higher levels of pepsin is used. Although there is no significant difference in the composition, flavor or texture of the cheese made with differing pepsin contents, the yield is 0.25% lower with pure bovine pepsin than is the case with rennet, an economically significant difference. In addition, pepsin has greater proteolytic activity than chymosin at all pH values from 2 to 6. If the proteolytic activity is excessive, the yield of cheese and retention of fat by the curd may be diminished. Excessive proteolysis during ripening also has undesirable effects on the body and flavor of the finished cheese. For these reasons, it is desirable to use rennin having a reduced pepsin level in the manufacture of cheese. Bovine pepsin can be inactivated at an alkaline pH for example, by incubating at a pH of 7-7.3 for 30 minutes at 30.degree. C. but not without inactivating some chymosin too. Because of the critical conditions necessary for the selective inactivation of pepsin, methods by which pepsin and chymosin are separated are to be preferred over the selective inactivation technique.
Garnot et al discuss the separation of chymosin from pepsin in calf rennet in J. Dairy Science, 55, 1641-1650 (1972). After exhaustive dialysis the sample is applied to a microgranular DEAE cellulose column. Both chymosin and pepsin are adsorbed on the column, however, application of a linear gradient of NaCl eluted chymosin first and pepsin at a higher salt concentration. This method was used primarily for the determination of the enzymatic composition of several commercial rennet preparations.
In Anal. Biochem., 139, 265-271 (1984), Pohl et al disclose the preparation of an affinity ligand (a synthetic inhibitor of aspartate proteinases) and its attachment to Sepharose 4B, which is then used to purify bovine cathepsin D and also human, porcine and chicken pepsins. These enzymes bind to the support over the pH range of 2-5. A buffer at pH.gtoreq.6, low ionic strength and containing 20% dioxane served as a general desorption agent. The peptide inhibitor (Sepharose) is a general purpose ligand which can bind most aspartate proteinases and is thus not discriminatory. Bovine Cathepsin D could be purified on the affinity column because the contaminant proteinases were all of the cysteine type (rather than aspartate proteinases) which did not bind to the column.
The use of pepstatin-Sepharose as the affinity medium for the purification of chymosin is disclosed in Agric. Biol. Chem., 42, 2227-2231 (1978) by Kobayashi et al. This method results in poor recovery (.apprxeq.60%) and pepstatin is quite expensive for use in commercial scale operations.
Dean et al in J. Chromatogr., 165, 301 (1979) and Burgett et al in Am. Lab., p. 74 (1977) enumerate a large number of enzymes which have successfully been separated on a Cibacron Blue F3GA column. These enzymes include hexokinase, DNA-polymerase, alcohol dehydrogenase, adenylate kinase, ribonuclease, glyoxalase, cytochrome C, aldolase, blood clotting factor X and enolase. These enzymes do not have many features in common either in terms of structure or function.